Process for producing dextrose using mixed immobilized enzymes

ABSTRACT

Process for converting starch to dextrose wherein a partially hydrolyzed starch solution containing at least 10 percent hydrolyzed starch is contacted with an enzyme system under conditions whereby substantially complete conversion of the starch to dextrose is achieved. The enzyme system comprises immobilized glucoamylase and alpha-amylase selected from the group consisting of soluble alpha-amylase, immobilized alpha-amylase and mixtures thereof.

This application is a continuation-in-part of U.S. Pat. application Ser.No. 500,975 filed Aug. 26, 1974, now U.S. Pat. No. 4,011,137.

THE INVENTION

This invention relates to a process for converting starch to dextrose.More particularly, this invention relates to a process for convertingstarch to dextrose by the use of an enzyme system comprising immobilizedglucoamylase and alpha-amylase selected from the group consisting ofsoluble alpha-amyalse, immobilized alpha-amylase and mixtures thereof.

Processes for hydrolyzing starch to dextrose are well known in the art.These methods can be grouped into two broad categories. These are theacid-enzyme and the enzyme-enzyme conversion processes. In theacid-enzyme process, generally, starch is first partially hydrolyzed orliquefied, for instance, by forming an aqueous suspension containingfrom 35 to 40 percent starch and incorporating therein an acid such ashydrochloric acid. The suspension is then heated to relatively hightemperatures to partially hydrolyze the starch and then cooled andtreated with a glucoamylase preparation under suitable conditions toenzymatically convert the partially hydrolyzed starch to dextrose. Theacid-enzyme process is disclosed, for example in U.S. Pat. Nos.2,304,168, 2,531,999, 2,893,921 and 3,042,584.

Glucoamylase has been referred to in the art as glucamylase glucogenicenzyme, starch glucogenase and gama-amylase. Glucoamylase is anexo-amylolytic enzyme which catalyzes the sequential hydrolysis ofglucose moieties from the non-reducing ends of starch or amylodextrinmolecules. Glucoamylase is elaborated by many types of microorganisms.Certain strains of fungi belonging to the Aspergillus group such asstrains belonging to the Aspergillus niger group and the Aspergillusawamori group, certain strains of the Rhizopus species and certainstrains of the Endomyces species elaborate glucoamylase.

In the enzyme-enzyme conversion process, generally, a starch slurry isformed and a starch liquefying enzyme, for instance, bacterialalpha-amylase, is added thereto and the starch slurry heated to atemperature in the range of 80° to 90° C. to partially hydrolyze thestarch. The partially hydrolyzed starch, which generally has a D.E. inthe range of from about 10 to 20, is then treated with glucoamylase.

Alpha-amylase is an endo-amylolytic enzyme capable of promoting almostrandom cleavage of α-1,4-glucosidic bonds within the starch molecule.Alpha-amylase is elaborated by many types of microorganisms such asmembers of the Bacillus subtilis species, Aspergillus niger and otherspecies of the Aspergillus genus and malted cereal grains.

Alpha-amylase will not act upon the α-1,6-glucosidic bonds in the starchmolecule to any significant degree. Glucoamylase will act upon suchbonds, but at a rate which is slower than is desired in commercialapplications.

Recently, there has been a great deal of interest shown in the use ofstarch debranching enzymes for dextrose production. The use of suchenzymes increases the amount of dextrose formed since they can readilyact upon bonds or linkages in the starch molecules which are not actedupon by alpha-amylase or which are only slowly acted upon byglucoamylase. Debranching enzymes are generally referred to asα-1,6-glucosidases. A number of enzymes having considerably differentspecificities have been identified in the art as being capable ofhydrolyzing α-1,6-glucosidic linkages. Of these, probably the two mostimportant from the commercial standpoint are pullulanase and isoamylase.The major difference in regard to the specificity of these enzymes isthat pullulanase will degrade the linear polysaccharide pullulan whereasisoamylase will not to any significant degree.

There are a number of patents which disclose methods of producingisoamylase and pullulanase and the utilization thereof. Canadian Pat.No. 852,196 to Ueda et al. describes a process for producing isoamylaseby cultivating a strain of Escherichia intermedia in a fermentationmedium comprising dextrins, peptone and inorganic salts. U.S. Pat. No.3,490,955 to Wallenfels et al. discloses a process for producingcellbound pullulanase from Aerobacter aerogenes in a culture mediumwherein the carbon sources comprise maltose and pullulan or glycerin.U.S. Pat. No. 3,560,345 to Yokobayashi et al. describes a process forproducing isoamylase by propagating Pseudomonas amyloderamosa in aculture medium containing as carbon sources, starch, starch derivativesor maltose.

Recently, there has been a great deal of interest shown in immobilizedenzymes. Immobilized enzymes have a number of distinct advantages oversoluble enzymes such as, for example, their use in continuous conversionsystems.

Exemplary of publications which review the art directed to enzymeimmobilization are the following:

Goldstein, in Fermentation Advances, Academic Press, New York, N.Y.(1969), pp. 391-424.

Goldstein et al., Z. Anal. Chem., 243, pp. 375-396 (1968).

Kay, Process Biochem., 3 (8), pp. 36-39 (1968).

Tosa et al., Kagaku To Seibutsu, 7 (3), pp. 147-155 (1967).

Silman et al., Ann. Rev. Biochem., 35 (2), pp. 873-908 (1966).

Gryszkiewicz, Folia Biologica, 19 (1), pp. 119-150 (1971).

Zaborsky, "Immobilized Enzymes", CRC Press, Cleveland, Ohio (1973).

In the art of enzyme immobilization, considerable interest has beendirected to the immobilization of glucoamylase. This is probably due tothe fact that in many commercial enzyme processes glucoamylase is usedin large amounts. The art is repleat with patents and publicationsdirected to immobilization of glucoamylase. Exemplary of such are thefollowing:

U.S. Pat. Nos. 2,717,852 to Stone; 3,619,371 to Crook et al.; 3,627,638to Barker et al.; 3,672,955 to Stanley; 3,715,277 to Dinelli et al.;Japanese Pat. Nos. 1360/60 and 23560/68; British Pat. Nos. 1,183,259 and1,183,260; German Pat. Nos. 2,062,246, 2,146,390 and 2,206,360.

Also: Usami et al., Hakko Kyokaishi, 25, pp. 513-516 (1967);

Barker et al., Carbohyd. Res., 9, pp. 257-263 (1969);

Wilson et al., Biotechnol. Bioeng., 11, pp. 349-362 (1969);

Usami et al., J. Ferment. Tech., 48, pp. 506-512 (1970);

Gruesbeck, Ph.D. Thesis, Univ. Texas (1970);

Bachler et al., Biotechnol. Bioeng., 12, pp. 85-92 (1970);

Maeda et al., Nippon Nogei Kagaku Kaishi, 44 (12), pp. 547-555 (1970);

Maeda et al., Hakko Kyokaishi, 28 (10), pp. 391-397 (1970);

Smiley, Biotechnol. Bioeng., 13, pp. 309-317 (1971);

Sorenson, MS Thesis, Purdue Univ. (1971);

Miyamoto et al., Hakko Kogaku Zasshi, 49 (6), pp. 565-573 (1971);

Usami et al., Hakko Kyokaishi, 29 (4), pp. 195-199 (1971);

O'Neill et al., Biotechnol. Bioeng., 13, pp. 337-352 (1971);

Emery et al., Chem. Eng. (London), No. 258, pp. 71-76 (1972);

Gruesbeck et al., Ind. Eng. Chem. Prod. Res. Develop., 11 (1), pp. 74-83(1972);

Beck, Ph.D. Thesis, Univ. Texas (1972);

Gestrelius et al., Biochem. Biophys. Acta, 276 (2), pp. 339-343 (1972);

Maeda et al., Agr. Biol. Chem., 36 (9), pp. 1581-1594 and pp. 1839-1842(1972);

Weetal et al., Biotechnol. Bioeng. Symp., No. 3, pp. 241-266 (1972);

Christison, Chem. & Ind. (London), (5), pp. 215-216 (1972);

Hough et al., Nature, 235, p. 389 (1972);

Corno et al., Die Staerke, 24, pp. 420-424 (1972);

Martensson et al., Biotechnol. Bioeng., 14 (5), pp. 715-724 (1972);

Park et al., J. Food Sci., 38, pp. 358-359 (1973).

There are also a number of patents and publications which disclose theimmobilization of alpha-amylase. Exemplary of such are the following:

U.S. Pat. Nos. 3,627,638 to Barker et al. and 3,715,278 to Miller;German Pat. Nos. 1,282,579, 1,943,490, 2,062,246 and 2,206,360.

Also: Grubhofer et al., Naturwissenschaften, 40, 508, (1953);

Manecke, Pure Appl. Chem., 4, pp. 507-520 (1962);

Manecke et al., Makromol. Chem., 51, pp. 199-216 (1962);

Bernfeld et al., Science, 142, pp. 678-679 (1963);

Manecke et al., Makromol. Chem., 91, pp. 136-154 (1966);

Fukushi et al., J. Biochem., 64, pp. 283-292 (1968);

Barker et al., Carbohyd. Res., 8, pp. 491-497 (1968);

Ledingham et al., Fed. Europ. Biochem. Soc. Lett., 5, pp. 118-120(1969);

Barker et al., Carbohyd. Res., 14, pp. 323-326 (1970);

Barker et al., Process Biochem., 5 (8), pp. 14-15 (1970);

Barker et al., Carbohyd. Res., 14, pp. 287-296 (1970);

Hough et al., Nature, 235, p. 389 (1972);

Epton et al., Carbohyd. Res., 22, pp. 301-306 (1972).

Additionally, there have been several patents and publications directedto processes for the immobilization of α-1,6-glucosidases. Exemplary ofsuch are the following:

British Pat. No. 1,258,095; Martensson et al., Biotechnol.

Bioeng., 14 (5), pp. 715-724 (1972).

From the above noted patents and publications, it is apparent that anumber of enzyme immobilization techniques have been described. Thesetechniques include covalently bonding an enzyme to a suitable insolublecarrier, encapsulation of an enzyme within a material which isimpermeable to the enzyme but permeable to the substrate and theproducts of the catalyzed reaction, adsorption of an enzyme on aninsoluble carrier and entrapment of an enzyme within a porous polymericmaterial wherein the pores are of such a size that will provide freeaccess of the substrate and the catalyzed reaction products but whichare sufficiently small to prevent the escape of the enzyme.

At low starch substrate concentrations, e.g., about 1 percent,glucoamylase preparations will substantially quantitatively convertunhydrolyzed starch to dextrose. Marshall et al., Fed. Europ. Biochem.Soc. Lett., 9 (2), pp. 85-88 (1970) and Fukui et al., Agr. Biol. Chem.,33 (6), pp. 884-891 (1969) reported that glucoamylase preparationsinherently contain alpha-amylase. When the alpha-amylase was removedfrom these preparations and the alpha-amylase-free glucoamylase was usedto saccharify a 1 percent starch solution, lesser amounts of dextrosewere formed than when glucoamylase preparations were used whichinherently contained alpha-amylase.

When a glucoamylase preparation is immobilized, the resultingimmobilized preparation is not capable of converting partiallyhydrolyzed starch to the same degree as the glucoamylase preparationfrom which the immobilized enzyme was prepared. Moreover, reactionscatalyzed by the immobilized glucoamylase preparation are not as rapidfor a given number of glucoamylase units used, especially during thelatter stages of the reaction period, as are reactions catalyzed by theglucoamylase preparation used for immobilization.

Therefore, it is the principal object of the present invention toprovide a method whereby substantially complete conversion of partiallyhydrolyzed starch to dextrose is achieved using an immobilized enzymesystem.

This object and other objects of the present invention which will beapparent from the following description are attained in accordance withthe present invention by contacting a partially hydrolyzed starchsolution containing at least 10 percent hydrolyzed starch with an enzymesystem comprising immobilized glucoamylase and alpha-amylase selectedfrom the group consisting of soluble alpha-amylase, immobilizedalpha-amylase and mixtures thereof under conditions wherebysubstantially complete conversion of the hydrolyzed starch to dextroseis achieved.

As mentioned above, when a glucoamylase preparation is subjected toimmobilization, the resulting immobilized glucoamylase does not convertpartially hydrolyzed starch so rapidly nor so completely as the solubleglucoamylase preparation from which the immobilized glucoamylase isprepared. We have found that during the immobilization of a glucoamylasepreparation, the alpha-amylase, which is inherently present therein, isrendered substantially inactive or inert regardless of the method ofimmobilization employed. This is surprising in view of the manydifferent methods that have been disclosed for the immobilization ofalpha-amylases. Although we do not wish to be bound to any theory, it isbelieved that the methods which have been found suitable for theimmobilization of glucoamylase are not suitable for the immobilizationof the alpha-amylase inherently contained in glucoamylase preparations.Apparently, the small amount of alpha-amylase which is inherentlypresent in soluble glucoamylase preparations has a beneficial effect onthe overall conversion of starch to dextrose with glucoamylase. Thus, toobtain maximum utilization of immobilized glucoamylase in the conversionof partially hydrolyzed starch to dextrose, there must also be presentduring the conversion soluble and/or immobilized alpha-amylase.Surprisingly, this finding is true even when the partially hydrolyzedstarch has been prepared by treatment of unmodified starch withalpha-amylase and therefore would be assumed to be rendered readilysusceptible to conversion with glucoamylase by such treatment. Moreover,it has been discovered that alpha-amylase added to immobilizedglucoamylase is effective for increasing the conversion of partiallyhydrolyzed starch to dextrose even during the latter stages of theconversion. Apparently, branched dextrins are formed during the initialstages of the hydrolysis reaction which are not readily hydrolyzed bythe immobilized glucoamylase but which are readily hydrolyzed byalpha-amylase and thus, the overall conversion of the starch hydrolysateis enhanced.

In the present process, the partially hydrolyzed starch may be preparedeither by an enzyme or acid treatment. In the case of enzyme treatment,the partially hydrolyzed starch should have a D.E. in the range of fromabout 10 to about 60. At substantially higher D.E. values, the amount ofdextrose formed will be limited due, presumably, to the presence ofsaccharides which are not readily acted upon by the immobilizedglucoamylase, while at lower D.E.s, the hydrolyzed starch has a tendencyto retrograde which includes the formation of a precipitate which maycoat the immobilized enzymes to such an extent that their efficiencywill be deleteriously affected. When a partial acid hydrolysate is usedin the present process, the D.E. thereof should be in the range of fromabout 10 to about 30. At higher D.E.s substantial amounts of reversionproducts are present which are not acted upon by the present enzymesystem.

The pH of the partial hydrolysate being treated may be in the range offrom about 3.5 to about 6.5 and preferably will be in the range of fromabout 4 to about 6.

The temperature of the partial hydrolyzate being treated in the presentprocess may vary relatively widely, but the temperature should not besufficiently high to inactivate the enzymes within a relatively shortperiod. Temperatures in the range of from about 30° to about 65° C. arepreferred and the most preferred temperatures are in the range of fromabout 50° to about 60° C. At these temperatures, the possibility ofundesirable microbial growth in the hydrolyzed starch is reduced andoptimum catalytic activity of the enzymes is generally obtained undernormal operating conditions.

The present process may be performed by a number of techniques. Forinstance, soluble or immobilized alpha-amylase and immobilizedglucoamylase may be used concurrently or sequentially. It is preferredthat they be used concurrently as, for example, when partiallyhydrolyzed starch is contacted with a mixture of immobilizedglucoamylase and immobilized alpha-amylase. Of course, it will berealized that the alpha-amylase and glucoamylase may be immobilized onor within the same carrier and results will be obtained which aresubstantially equivalent to those given by mixtures of alpha-amylase andglucoamylase immobilized on separate carriers. In the case where theenzymes are used sequentially, the conversion process will comprise atleast three steps in the following sequence: (1) contacting the partialhydrolysate with immobilized glucoamylase, (2) contacting the resultinghydrolysate with a soluble or immobilized alpha-amylase, and (3)contacting the resulting hydrolysate with immobilized glucoamylase. Thelast two steps of the sequence may be repeated a number of timesdepending on the conditions under which the reactions are conducted. Theconcurrent use of the enzymes results in greater amounts of thepartially hydrolyzed starch being converted to dextrose than doessequential use except when the steps employed in sequential use arerepeated a large number of times.

Alpha-amylase and glucoamylase may be immobilized by entrapment within apolymeric matrix. In the entrapment process, an insoluble, threedimensional polymeric matrix is formed with the enzyme dispersedthroughout. The pores of the polymeric matrix are too small to allow theenzyme to escape, but are large enough to permit diffusion of substrateand product molecules in and out. Once the matrix is formed in theentrapment procedure, the product so formed may be treated with agentsto bond the enzyme to the matrix and/or to strengthen or furtherinsolubilize the matrix as is described, for example, in U.S. Pat. No.3,838,007 to Van Velzen. The enzyme may also be made a part of thepolymeric matrix as, for example, when a mixture comprising the enzymeand other polymers is treated with a cross-linking agent resulting in aninsoluble gel.

Entrapment of enzymes is distinct from encapsulation of enzymes. In thelatter, enzyme is enveloped within a wall comprising a thin polymericfilm but is not dispersed within the polymer network comprising the filmas in the entrapment process. Thus, entrapment of enzymes is distinctfrom encapsulation of enzymes as disclosed in U.S. Pat. No. 3,849,253 toHarvey.

A number of different types of alpha-amylase may be used, although it ispreferred that saccharifying or pancreatic type alpha-amylase be used.Microorganisms such as Bacillus subtilis var. amylosacchariticusFukumoto elaborate saccharifying type alpha-amylase. Generally, it isalso preferred that alpha-amylase preparations which are to be used forimmobilization have an S/L value (hereinafter defined) of at least about3, preferably at least about 50 and most preferably a value of at leastabout 100.

The ratio of the activities of the enzymes used in the present processshould typically be above a certain minimum value to provide optimumcatalytic action. In this regard, the amounts of immobilizedglucoamylase and of alpha-amylase which may be used should be sufficientto provide a ratio of dextrinizing activity (hereinafter defined) toglucoamylase activity (hereinafter defined) of at least 0.2 liquefonsper glucoamylase unit. Preferably, the amounts of enzymes present willbe sufficient to provide at least 1 liquefon per glucoamylase unit, andmost preferably, the amounts will be sufficient to provide at least 3liquefons per glucoamylase unit.

When the present enzyme system is used in a column or bed, or in othermeans whereby such can be used in a continuous manner, it is importantto remove any insoluble material which may be present in the partialstarch hydrolysate so that such material does not plug the column orcoat the immobilized enzymes to a degree which substantially reduces theefficiency of the enzyme system. Removal of insoluble material may beaccomplished in any convenient manner such as filtration, centrifugationor the like.

Immobilized α-1,6-glucosidases may also be used in the present process.Exemplary of the preferred enzyme of this class is pullulanase. It ispreferred to immobilize the pullulanase by covalently binding it to aninert carrier.

In order to more clearly describe the nature of the present invention,specific examples will hereinafter be described. It should beunderstood, however, that this is done solely by way of example and isintended to neither delineate the scope of the invention nor limit theambit of the appended claims.

Expressions and procedures referred to in the present specification andclaims are defined below:

Dextrose Equivalent

Dextrose equivalent (D.E.) was determined by Method E-26 described in"Standard Analytical Methods of the Member Companies of the CornIndustries Research Foundation", Corn Refiners Association, Inc., 1001Connecticut Avenue, N.W., Washington, D.C. 20036.

Dextrose Content

Dextrose content was calculated from the Mathews' Index. For adiscussion of Mathews' Index see Cayle and Viebrock, Cereal Chem., 43,237 (1966).

The Mathews' Index was determined from measurements of optical rotationand reducing sugar content of the converted solutions. The convertedsolutions were diluted to about 3 percent dry solids and opticalrotation (R) determined in degrees circular in a 0.2-dm., jacketed cellmaintained at 25° C. using an automatic polarimeter (Bendix ScientificInstruments, Model NPL) equipped with a green light source (546.1 nm). Aportion of the solution used for polarimetry was diluted four fold andtitrated into 25 ml of Fehling's solution according to the method fordetermining D.E. enumerated above. The titre (T) so obtained is thenumber of mls of diluted solution which contains reducing sugarsequivalent to 0.12 g of dextrose. The Mathews' Index (M) was calculatedfrom the rotation (R) and titre (T) as follows:

    M = RT/4

percent dextrose (ash free, dry substance basis) was then calculatedfrom the Mathews' Index by the following equation:

    Percent Dextrose = (170 - 20 M)/(0.2167 M + 1.0784)

Preparation of Partially Hydrolyzed Starch Solution

The partially hydrolyzed starch solutions used in the various analyticaldeterminations and in the following examples were prepared using thefollowing general procedure:

An 18° Be slurry of corn starch in water was adjusted to pH 7.0 withlime and alpha-amylase (B. subtilis origin, 33 liquefons per g drystarch) added. The mixture was instantaneously heated to 88° C. togelatinize the starch and initiate enzyme action by blending with steamin a mixing jet and was then held at 88° C. for about one hour. Themixture was then heated to 149° C. by blending with steam under pressurein a mixing jet, held at 149° C. for about one minute and then cooled to88° C. in a vacuum chamber. Additional alpha-amylase (11 liquefons per gof dry starch) was added to the mixture at 88° C. and hydrolysiscontinued until the desired D.E. was obtained. After cooling to 60° C.,the solution was adjusted to pH 3.5 to 4 using 4 M hydrochloric acid andwas then heated for 90 minutes at 100° C. to inactivate any residualalpha-amylase activity. Three percent filter aid was added and the hothydrolyzate was filtered to remove insoluble protein and fat. The aboveprocedure provided partially hydrolyzed starch solution having a D.E. of12 to 20 and 31 to 34 percent dry solids.

Glucoamylase Activity

A glucoamylase activity unit (GU) is defined as the amount of enzymewhich catalyzes the production of one g of dextrose per hour at 60° C.at pH 4.5 in the procedures described below.

Drum-dried partially hydrolyzed starch was used for the preparation ofsubstrate solutions for glucoamylase activity determinations. Apartially hydrolyzed starch solution having a D.E. of 12, was treatedwith activated carbon (Nuchar CEE, West Virginia Pulp and Paper Co.) for45 minutes at 60° C. The carbon was removed by filtration and thefiltrate was treated again with carbon and filtered in the same manner.The filtrate was concentrated to about 50 percent dry solids and wasthen dried on a steam-heated drum drier and ground. The drum-driedpartially hydrolyzed starch contained 1.7 percent moisture and 0.5percent ash. Substrate solutions for glucoamylase activitydeterminations were prepared to contain 10 g of the dried hydrolyzedstarch and 2 ml of pH 4.5, 1 M sodium acetate buffer per 100 ml ofsolution.

Activity of Soluble Glucoamylase

Ten ml of substrate solution was pipetted into a capped reactormaintained at 60° C. One ml of glucoamylase solution containing 0.03 to0.15 GU was added and mixed therein and the mixture maintained for onehour at 60° C. At the end of the 1-hour incubation period, enzyme actionwas stopped by adding a predetermined volume of 1 M sodium hydroxidesolution so as to obtain a pH of 8.5 to 10.5. The mixture was thencooled to room temperature.

2.5 ml of the assay hydrolysate so obtained was pipetted into 25 ml ofFehling's solution prepared as described in the above cited method forD.E. determination. The mixture was brought to a boil and titrated withstandard dextrose solution containing 5 g of dextrose per literaccording to the procedure cited above for D.E. determination. A controlmixture was prepared and titrated in the exact same manner as for theassay hydrolysate above except that the 1 ml of glucoamylase solutionwas added to the substrate solution after the one-hour incubation periodand after the addition of sodium hydroxide solution. Glucoamylaseactivity was calculated as follows:

    GU/ml = 0.002 V (C-A)

where V is the total volume (ml) of assay hydrolysate (usually 11.2 ml),C is the ml of standard dextrose solution used in the titration of thecontrol mixture, and V is the ml of standard dextrose solution used intitration of the assay hydrolysate.

Activity of Immobilized Glucoamylase

The activity of immobilized glucoamylase was determined by amodification of the above procedure for determining the activity ofsoluble glucoamylase. 10 ml of substrate solution prepared as describedabove was heated in a closed reactor to 60° C. A weighed amount (W) ofimmobilized glucoamylase containing from 3 to 10 GU was suspended indemineralized water and was diluted to 100 ml. The immobilizedglucoamylase suspension was stirred and while stirring, a 1-ml aliquotof the suspension was transferred to the 10 ml of the substrate solutionheld at 60° C. The mixture was stirred continually for exactly 1 hour at60° C. and was then filtered to remove the immobilized glucoamylase. 2.5ml of the assay filtrate so obtained was added to 25 ml of Fehling'ssolution and titrated with standard dextrose in the manner describedabove for determining the activity of soluble glucoamylase. A controlfiltrate was prepared and titrated by the exact same steps except thatone ml of water was substituted for the one ml of immobilizedglucoamylase suspension. Immobilized glucoamylase activity wascalculated as follows:

    GU/g = 2.2 (C.sub.i - A.sub.i)/W

where C_(i) is the ml of standard dextrose solution used in thetitration of the control filtrate, A_(i) is the ml of standard dextrosesolution used in the titration of the assay filtrate and W is the weight(g) of immobilized glucoamylase in the 100 ml of suspension.

Activity of Alpha-Amylase

Alpha-amylase preparations were assayed by two different methods. In onemethod, the ability of the alpha-amylase preparation to hydrolyzesoluble Lintner starch to dextrins too small to give a blue color withiodine was determined as a measure of dextrinizing activity. In theother method, the ability of the alpha-amylase preparation to producereducing sugars by the hydrolysis of a reduced partially hydrolyzedstarch was determined as a measure of saccharifying activity.

Dextrinizing Activity of Soluble Alpha-Amylase

The dextrinizing activity of soluble alpha-amylase preparations wasdetermined by a modification of Standard Test Method, AATCC 103, 1965,"Bacterial Alpha-Amylase Enzymes Used in Desizing, Assay of" publishedin the 1967 Edition of Technical Manual of the American Association ofTextile Chemists and Colorists, Volume 43, pp. B-174 and B-175. Themethod was modified by substituting 10 ml of 1 M sodium acetate buffer,pH 5.0, for the 10 ml of pH 6.6 phosphate buffer solution used in themakeup of the buffered starch substrate. Also, 0.73 g of CaCl₂.2H₂ O wasadded per 500 ml of buffered starch substrate. Results were calculatedin terms of liquefons where one liquefon equals 0.35 Bacterial AmylaseUnit.

Dextrinizing Activity of Immobilized Alpha-Amylase

The dextrinizing activity of immobilized alpha-amylase preparations wasdetermined in the same manner as for soluble alpha-amylase preparationsexcept that immobilized alpha-amylase was "diluted" for assay bysuspension in 0.005 M calcium acetate solution at 30° C. A 5-ml aliquotof the suspension was added to the 10 ml of buffered starch substrateand the hydrolyzing mixture so formed was stirred continuously duringthe 30° C. hydrolysis step. At appropriate time intervals, 2-ml aliquotsof the hydrolyzing mixture were taken and rapidly filtered and one ml ofthe filtrate added to the 5 ml of dilute iodine solution. Time wascounted starting at the instant the 5-ml aliquot of suspension was addedto the 10 ml of buffered starch substrate and finishing at the time thatthe 2-ml aliquot of hydrolyzing mixture was filtered.

Saccharifying Activity of Soluble Alpha-Amylase

Saccharifying activity of soluble alpha-amylase preparations wasdetermined using a reduced partially hydrolyzed starch solution (RLS) asa substrate. One unit of saccharifying activity (S) was defined as theamount of enzyme which would produce an increase of 0.02 absorbance unitper minute in the procedure described below.

The RLS was prepared from a 1.8-liter sample of 12 D.E. partiallyhydrolyzed starch solution containing 31 percent dry solids. Thepartially hydrolyzed starch solution was prepared as describedpreviously except that the final steps in its preparation comprisingadjustment of the pH to 3.5 to 4.0 and heating to 90° C. to inactivateresidual alpha-amylase and filtration were omitted. The hydrolyzedstarch solution was adjusted to pH 6.5 to 7.0 and was heated to 70° C.Liquefying alpha-amylase preparation of B. subtilis origin containing57,000 liquefons was added and the mixture held 3.5 hours at 70° C. ThepH was adjusted to 3.5 to 4.0 and the mixture heated one hour at 100° C.3 percent filter aid was added and the mixture was filtered. Thefiltrate was adjusted to pH 5.5 with 8 M NaOH solution, 10 g ofDEAE-cellulose (Whatman DE 23, Reeve Angel) added, and the mixturestirred for 30 minutes at ambient temperature. The mixture was thenmaintained for 18 hours at 5° C. without stirring, heated to 60° C. andfiltered. The filtrate was refined twice by stirring for 60 minutes at60° C. with 16 g of activated carbon (Nuchar CEE) and about 50 g offilter aid followed by filtering. 975 ml of the twice-refined filtratewas obtained containing 34.5 percent dry solids and having a D.E. of30.2. 400 ml of the twice-refined filtrate was cooled to about 20° C.and 0.5-g portions of sodium borohydride dissolved therein at 30-minuteintervals until 6.0 g had been added. The resulting solution was stirredat ambient temperature for about 16 hours, was recooled to about 2° C.and 0.5 g of sodium borohydride dissolved therein. After stirring for 4hours, a final 0.5-g portion of sodium borohydride was added and thesolution stirred for 26 hours at ambient temperature. 435 ml of theresulting solution was refined by charging the same to an ion-exchangecolumn containing an 89 × 2.5-cm bed of Borosorb (Calbiochem, CN 203667)and washing the charge through the column with water. The effluent(charge plus washings) was concentrated to 800 ml and then was chargedto three columns placed in series as follows: an 89 × 2.5-cm bed ofBorosorb, a 26 × 2.5-cm bed of strong acid resin in hydrogen form(Duolite C-3, Diamond Shamrock Chemical Co.), and a 62 × 2.5-cm bed ofweak base resin in free amine form (Duolite A-6). The charge was washedthrough the columns with water and the effluent collected until 4.9liters had been recovered. The effluent was concentrated to 450 ml, 0.09g of sodium azide added and the mixture filtered through a membranefilter (Nalge Corp.) having a maximum pore size of 0.2 microns. The RLSsolution so prepared had the following properties: D.E. less than 0.4,pH 6.4, 26.3 percent dry solids, 0.03 percent sulfated ash, 1.7 ppmboron.

An RLS substrate solution for measuring saccharifying activity wasprepared to contain 2 g dry basis RLS, 2 ml of 1 M, pH 5.0 sodiumacetate buffer, and 0.147 g of CaCl₂.2H₂ O in a total volume of 100 ml.For the saccharifying activity determination, 5 ml of RLS substratesolution equilibrated to 30° C. was mixed with 5 ml of a solution of thealpha-amylase preparation diluted to contain 0.2 to 1.0 S/mlequilibrated to 30° C. The hydrolyzing mixture was incubated at 30° C.and 1-ml aliquots removed at 1, 3 and 5 minutes after combination of theenzyme and substrate solutions. Each aliquot was immediately combinedwith 1 ml of dinitrosalicylic acid reagent prepared according to P.Bernfeld in "Methods in Enzymology", S. P. Colowick and N. O. Kaplan,editors, Vol. I, p. 149, Academic Press, New York (1955). The mixturewas heated for 5 minutes in a boiling water bath and was then cooled forat least 10 minutes in cold, running tap water (about 15° C.). Themixture was diluted by adding 10 ml of demineralized water and theabsorbance of the resulting solution determined at 450 nm in a 1-cmcell. A plot of absorbance vs. incubation time was made and the slope(Y) of the plot determined in absorbance units per minute. The activityof the diluted solution of alpha-amylase preparation was calculated asfollows:

    Activity (S/ml) = 10 Y

s/l value

The soluble alpha-amylase preparations derived from different sourcesand used for the preparation of immobilized alpha-amylase wereclassified by their S/L Value which was defined as one thousand timesthe saccharifying activity measured in saccharifying units (S) per g ofalpha-amylase preparation divided by the dextrinizing activity measuredin liquefons per g of preparation.

Activity of Soluble Pullulanase Preparations

Pullulanase activity was determined by its hydrolytic effect on pullulanusing an alkaline ferricyanide reagent to determine the maltotrioseliberated. Activity was expressed in international units (IU) where oneIU is the amount of pullulanase which catalyzes the liberation of 1micromole of maltotriose per minute from a 0.5 percent solution ofpullulan at pH 5.0 and 45° C.

The ferricyanide reagent was prepared by dissolving 0.85 g of potassiumferricyanide and 10 g of sodium carbonate in demineralized water anddiluting to one liter. The reagent was calibrated against solutions ofmaltotriose (Pierce Chemical Co.). 2-ml aliquots of ferricyanide reagentwere mixed in test tubes with 1-ml aliquots of maltotriose solutionscontaining 25, 100, 150, 200 or 250 micrograms of maltotriose per ml.The tubes were immersed in a boiling water bath for 10 minutes and thencooled for 10 minutes at ambient temperature and absorbance measured ina 1-cm cell at 420 nm. Maltotriose concentration was plotted versusabsorbance and a calibration factor (C) determined from the slope of theplot.

To determine pullulanase activity, a test tube containing 9.5 ml ofsubstrate solution comprising 8.5 ml of 0.02 M, pH 5.0 sodium acetatebuffer and 1.0 ml of a solution containing 50 mg of pullulan wasincubated in a 45° C. water bath for 5 minutes. A 0.5-ml aliquot ofpullulanase solution was added to the test tube and mixed therein. At 5,10, 15, and 20 minutes after the addition of the pullulanase solution,1.0-ml aliquots of the reacting mixture were pipetted into test tubescontaining 2 ml of the above calibrated ferricyanide reagent. Themixtures were heated, cooled, and their absorbances determined as forthe calibration of the ferricyanide reagent above. Absorbance versustime was plotted and the slope (K) of the rate plot determined. Activityof the pullulanase solution was calculated from the following formula:

    Activity (IU/ml) = 0.0397 CK

where C is the ferricyanide reagent calibration factor (micrograms ofmaltotriose per ml per absorbance unit) and K is the slope of the rateplot (absorbance units per minute).

Activity of Immobilized Pullulanase Preparation

Activity of the immobilized pullulanase preparation was determined bythe method described above for soluble pullulanase with the followingexceptions. A suspension was formed by stirring 25 mg of immobilizedpullulanase preparation in 5.0 ml of demineralized water and a 0.5-mlaliquot of the suspension added to the 9.5 ml of substrate solution toform the reaction mixture. The reaction mixture was stirred constantlyduring the reaction period. Aliquots were withdrawn at 5, 10, 15 and 20minutes, were quickly filtered, and 1.0-ml portions combined with 2-mlaliquots of the ferricyanide reagent.

EXAMPLE I

This example illustrates the use of glucoamylase immobilized onDEAE-cellulose and alpha-amylase derived from different sourcesimmobilized on aminoethyl-cellulose for converting a partiallyhydrolyzed starch solution to dextrose.

Immobilization of Glucoamylase

53.0 g of a dry glucoamylase preparation (from Aspergillus awamori, freeof transglucosylase activity) having a glucoamylase activity of 83.2 GUg⁻¹ was incorporated into 3.8 liters of deionized water. The mixture wasstirred for 30 minutes and filter aid added thereto. The mixture wasfiltered, the filter cake washed, the filtrate and washings combined,and the pH of the combined solutions adjusted to 5.5 using 4 N HCl. 13.3g DEAE-cellulose (Whatman DE 23) was added, the mixture stirred for 60minutes at ambient temperature and then filtered and the filter cakewashed with deionized water. The recovered moist filter cake had aglucoamylase activity of 44 GU g⁻¹. The moist filter cake is hereinafterreferred to in this example as "immobilized glucoamylase".

Immobilization of Alpha-Amylase

Alpha-amylase derived from various sources was immobilized by couplingthe alpha-amylase with activated aminoethyl-cellulose (hereinafterreferred to as "activated AE").

The activated AE was prepared by slurrying 20 g of aminoethyl-cellulose(Cellex-AE manufactured by Bio-Rad Laboratories) in 500 ml of a 0.5 Mphosphate buffer at pH 7, stirring for 20 minutes at ambient temperatureand then maintaining the mixture for 7 hours without stirring. Themixture was filtered, the filter cake washed with deionized water andsuspended for 12 hours in 500 ml of 0.5 M phosphate buffer at pH 7. 140ml of a glutaraldehyde solution (50 percent) was added to the slurry,the slurry stirred for 90 minutes at ambient temperature, filtered andthe filter cake washed with deionized water. 74.9 g of filter cake (75.2percent moisture) was recovered.

16.0-g portions of activated AE were added to 50 ml of each of thefollowing four alpha-amylase solutions:

(a) Solution of Bacillus subtilis saccharifying alpha-amylase (var.amylosacchariticus Fukumoto, twice recrystallized, Miles Laboratories,Inc., S/L = 257) containing 0.33 mg protein per ml and having anactivity of 134 liquefons per ml.

(b) Solution of Bacillus subtilis liquefying alpha-amylase (BacterialType, II-A, 4x crystallized, Sigma Chemical Co., S/L = 5) containing0.45 mg protein per ml and having an activity of 1166 liquefons per ml.

(c) Solution of Aspergillus oryzae fungal alpha-amylase (3xcrystallized, Calbiochem, S/L = 76) containing 0.45 mg protein per mland having an activity of 322 liquefons per ml.

(d) Solution of hog pancreatic alpha-amylase (2x crystallized,Worthington Biochemical Corp., S/L = 227) containing 0.70 mg protein perml and having an activity of 290 liquefons per ml.

The mixtures were stirred for 2 hours and filtered. The filter cakeswere washed with 0.005 M calcium acetate at pH 7 with small portions of0.5 M NaCl (total 100 ml) and then with about 50 ml of 0.05 M calciumacetate solution. The immobilized alpha-amylases exhibited the followingpotencies:

    ______________________________________                                                            Potency                                                   Alpha-Amylase       (liquefons g.sup.-1)                                      ______________________________________                                        Immobilized saccharifying                                                                         12.6                                                      Immobilized liquefying                                                                            125.0                                                     Immobilized fungal  7.3                                                       Immobilized pancreatic                                                                            41.5                                                      ______________________________________                                    

To each of five stirred reactors maintained in a water bath at 50° C.was added 138 g of partially hydrolyzed starch solution (pH 5.0, 16.9D.E., 32.5 percent dry substance) prepared by the procedure describedabove which had been filtered through a cellulose ester membrane (HAWP04700, 0.45 μ, Millipore Corp.) and then saturated with toluene. Thepurpose of the addition of the toluene was to prevent bacterial growth.

0.51 g of immobilized glucoamylase was added to each of the reactors andsufficient immobilized alpha-amylase was added to four of the reactorsto provide a total of 90 liquefons of alpha-amylase activity perreactor. The reactors were continually stirred and at various timeintervals, samples were taken from the reactors and filtered and thefiltrates assayed for percent dextrose. The results of this example areset forth in Table I.

                                      TABLE I                                     __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase and Various Types of          Immobilized                                                                   Alpha-Amylase                                                                                Average pH of system                                                                      Percent Dextrose                                   Immobilized Enzyme System                                                                    During Saccharification                                                                   46 hrs                                                                            70 hrs                                                                            106 hrs                                                                           142 hrs                                __________________________________________________________________________    Immobilized glucoamylase                                                                     5.1         71.5                                                                              76.3                                                                              79.6                                                                              80.9                                     (control)                                                                   Immobilized glucoamylase                                                                     5.1         92.3                                                                              92.9                                                                              93.0                                                                              92.7                                   and immobilized saccharifying                                                 alpha-amylase                                                                 Immobilized glucoamylase                                                                     5.0         89.0                                                                              91.5                                                                              93.1                                                                              93.2                                   and immobilized liquefying                                                    alpha-amylase                                                                 Immobilized glucoamylase                                                                     5.2         87.2                                                                              89.7                                                                              91.5                                                                              91.9                                   and immobilized fungal                                                        alpha-amylase                                                                 Immobilized glucoamylase                                                                     5.1         93.1                                                                              94.7                                                                              94.9                                                                              94.2                                   and immobilized pancreatic                                                    alpha-amylase                                                                 __________________________________________________________________________

From the above table, it is seen that a combination of immobilizedglucoamylase and immobilized alpha-amylase resulted in a more completeconversion of starch to dextrose than when immobilized glucoamylasealone was used. Also, the combination of immobilized enzymes resulted ina more rapid conversion of starch to dextrose. Moreover, in general, theimmobilized alpha-amylase preparations prepared from the solublealpha-amylase preparation having a high S/L ratio are more beneficial inthe conversion of partially hydrolyzed starch to dextrose.

EXAMPLE II

This example illustrates the effect of the ratio of immobilizedalpha-amylase activity to immobilized glucoamylase activity on the rateof production of dextrose.

60 g of activated AE prepared according to Example I was added to 975 mlof 0.005 M sodium acetate solution containing 5 × 10⁵ liquefons offungal alpha-amylase preparation derived from Aspergillus oryzae(Enzeco® K768, Enzyme Development Corp.). After slurrying for two hoursat ambient temperature, the slurry was filtered, and the filter cakewashed successively with deionized water, one liter of partiallyhydrolyzed starch solution (3.2 percent d.s., 16.4 D.E., pH 5.0, 0.02percent NaN₃) and 200 ml of 0.02 percent NaN₃ solution. The moist filtercake had an alpha-amylase activity of 33.3 liquefons g⁻¹.

Saccharification Using Immobilized Glucoamylase and ImmobilizedAlpha-Amylase Derived from Aspergillus oryzae.

Into 6 stirred reactors each containing 462 g of partially hydrolyzedstarch solution (32.0 percent d.s., 16.7 D.E., pH 5.1, 0.02 percentNaN₃) at 50° C. was added 1.13 g of immobilized glucoamylase preparedaccording to the method described in Example I and having a potency of64.2 GU g⁻¹. Then into the reactors were added, respectively, 21.8 g,10.9 g, 5.45 g, 2.72 g, 1.36 g and 0 g of immobilized fungalalpha-amylase prepared by the procedure described immediately above. Thereactors were constantly stirred at 50° C. and the percent dextrosedetermined at various periods.

Saccharification Using Immobilized Glucoamylase and ImmobilizedAlpha-Amylase of the Pancreatic Type.

Into 6 stirred reactors each containing 121 g of partially hydrolyzedstarch solution (33.4 percent d.s., 16.9 D.E., pH 5.1, saturated withtoluene) at 50° C. was added 0.45 g of immobilized glucoamylase preparedaccording to Example I and having a potency of 44 GU g⁻¹. Then into thereactors were added, respectively, 4.82 g, 2.41 g, 1.20 g, 0.60 g, 0.30g and 0 g of immobilized pancreatic alpha-amylase prepared by theprocedure set forth in Example I. The reactors were constantly stirredat 50° C. and the percent dextrose determined at various periods.

The results of these experiments are shown below in Tables II and III:

                                      TABLE II                                    __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase and Immobilized Fungal        Alpha-Amylase                                                                 Ratio of Alpha-Amylase Activity to                                            Glucoamylase Activity (liquefons                                                                 Percent Dextrose                                           per GU)*           20 hr                                                                             44 hr                                                                             68 hr                                                                             92 hr                                                                             116 hr                                                                            140 hr                                 __________________________________________________________________________    0 (Control)        63.6                                                                              74.9                                                                              79.1                                                                              81.9                                                                              83.2                                                                              84.7                                   0.62               70.0                                                                              80.7                                                                              84.8                                                                              86.7                                                                              87.5                                                                              89.1                                   1.25               73.0                                                                              83.1                                                                              86.4                                                                              88.2                                                                              89.1                                                                              90.4                                   2.5                76.2                                                                              85.2                                                                              88.0                                                                              89.9                                                                              90.5                                                                              91.5                                   5.0                78.6                                                                              86.8                                                                              89.2                                                                              90.6                                                                              91.2                                                                              92.4                                   10.0               80.8                                                                              88.8                                                                              90.7                                                                              92.1                                                                              92.2                                                                              92.7                                   __________________________________________________________________________     *Each reactor contained 0.5 GU g.sup.-1 partially hydrolyzed starch      

                                      TABLE III                                   __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase and Immobilized               Pancreatic                                                                    Alpha-Amylase                                                                 Ratio of Alpha-Amylase Activity to                                            Glucoamylase Activity (liquefons                                                                 Percent Dextrose                                           per GU)*           20 hr                                                                             44 hr                                                                             68 hr                                                                             92 hr                                                                             116 hr                                                                            140 hr                                 __________________________________________________________________________    0 (Control)        62.1                                                                              72.3                                                                              77.1                                                                              80.1                                                                              81.6                                                                              82.5                                   0.62               69.2                                                                              82.7                                                                              87.7                                                                              90.1                                                                              90.8                                                                              91.2                                   1.25               77.2                                                                              88.4                                                                              92.0                                                                              93.1                                                                              93.7                                                                              93.4                                   2.5                81.8                                                                              91.2                                                                              93.4                                                                              94.3                                                                              94.3                                                                              94.1                                   5.0                85.1                                                                              92.9                                                                              94.6                                                                              95.2                                                                              95.2                                                                              94.7                                   10.0               87.6                                                                              94.0                                                                              95.2                                                                              95.6                                                                              95.1                                                                              95.6                                   __________________________________________________________________________     *Each reactor contained 0.5 GU g.sup.-1 partially hydrolyzed starch      

From the above tables it is seen that as the ratio of alpha-amylaseactivity to glucoamylase activity is increased, the faster and the morecomplete is the conversion. However, at the higher ratios the differencein the conversion is small which indicates that there is a maximum ratiowhere no substantial increase in conversion is obtained.

EXAMPLE III

This example illustrates the utilization of covalently immobilizedglucoamylase and various other immobilized enzymes.

Immobilization of Glucoamylase

20 g of DEAE-cellulose (Whatman DE 23) was slurried in 500 ml of 1NNaOH, stirred for 30 minutes at ambient temperature and the slurryfiltered. The filter cake was slurried in 30 ml of acetone containing4.0 g cyanuric chloride for one minute and then 600 ml of 20 percentacetic acid solution was added. After about one minute, the slurry wasfiltered, the filter cake washed with deionized water and suspended in800 ml of a 50 percent (v/v) mixture of 0.2 Mtris(hydroxymethyl)aminomethane and 5N HCl. After stirring for 7minutes, 600 ml of 20 percent acetic acid was added to the slurry andstirring continued for another minute. The slurry was filtered, thefilter cake washed extensively with deionized water and then with 500 mlof acetone. The filter cake was dried by applying partial vacuumthereto. 19 g of filter cake was recovered.

The filter cake was added to 2000 ml of a glucoamylase solution (3.8 GUml⁻¹) free of transglucosylase prepared by thoroughly dialyzing aglucoamylase concentrate against tap water and then against pH 8.1borate buffer (0.05 M). After stirring for 20 hours at ambienttemperature, the slurry was filtered and the filter cake washedextensively with deionized water. The moist filter cake was thensuspended in 500 ml of 1M NaCl, stirred 30 minutes and filtered. Thefilter cake was washed with 500 ml of a 1M NaCl solution and then withdeionized water. The filter cake weighed 74.3 g and had an activity of15 GU g⁻¹.

Immobilization of Pullulanase

Aerobacter aerogenes ATCC 15050 was propagated and the pH of thefermentation broth was adjusted to 7 by the addition of a 0.2 M solutionof NaH₂ PO₄. 80 g of Triton X-100 (Rohm & Haas) was also added to thebroth. The broth was stirred for 16 hours at 35° C., centrifuged at18,000 × g for 10 minutes and the sediment discarded. The supernate hada pullulanase activity of 1.07 IU ml⁻¹.

The pH of a 4000-ml portion of the supernate was adjusted to 7.6 by theaddition of a solution of 0.2 M Na₂ HPO₄ and 6 g of DEAE-Sephadex A-50(Pharmacia) was added. The slurry was stirred for 30 minutes at ambienttemperature, filtered and the filter cake washed with 1000 ml ofdeionized water. The filter cake was suspended in 100 ml of pH 7.0, 0.01M phosphate buffer containing 5.4 g NaCl and stirred for 30 minutes. Theslurry was filtered and the filtrate concentrated to 290 ml byultrafiltration in an Amicon model 401 ultrafiltration cell equippedwith an XM-50 membrane. This filtrate was then dialyzed againstdeionized water to obtain a solution having a pullulanase activity of10.5 IU ml⁻¹.

80 g of Whatman standard grade powdered cellulose was suspended in 500ml of 5 M NaOH solution and allowed to stand for 16 hours. The supernatewas removed by decantation and the cellulose washed several times withdeionized water. The supernate was again removed by decantation and thecellulose filtered and suspended in 500 ml of deionized water. A 200 mlaliquot of the suspension containing about 20 g dry basis cellulose wasadjusted to pH 10.5 by the addition of 1 M NaOH solution. 50 ml of asolution containing 5 g of cyanogen bromide was added and during a45-minute reaction period, the pH of the mixture was maintained in therange of from 10.0 to 10.5 by the periodic addition of 1M NaOH solution.The cyanogen bromide-activated cellulose was collected by filtration,the filter cake washed with 1000 ml of deionized water and then with 200ml of 0.01 M sodium phosphate buffer at pH 7.9.

200 ml of the dialyzed filtrate having a pullulanase activity of 10.5 IUml⁻¹ was adjusted to pH 7.9 by the addition of 0.2 M Na₂ HPO₄ solution.10 g of the cyanogen bromide-activated cellulose was added, thesuspension stirred for 16 hours at a temperature of about 3° C. andfiltered, and the filter cake washed with 50 ml of 1 M NaCl solution.The washed filter cake had a pullulanase activity of 80.2 IU g⁻¹.

Immobilization of Saccharifying Alpha-Amylase

7 g of cyanogen bromide-activated cellulose (prepared by the proceduredescribed above) was added to 99 ml of cold 0.1 M phosphate buffer at pH8 having dissolved therein sufficient saccharifying alpha-amylase (B.subtilis var. amylosacchariticus) to obtain an activity of 260 liquefonsml⁻¹. The suspension was stirred for 20 hours while being maintained at5° C. and was filtered, the filter cake washed, successively, with 0.1 Mphosphate buffer, with deionized water, with 2 percent Lintner starchsolution at pH 5 and finally again with deionized water.

The washed filter cake had an alpha-amylase activity of 74 liquefonsg⁻¹.

Utilization of the Immobilized Enzymes

Four stirred reactors each containing 400 ml of partially hydrolyzedstarch solution (25.6 percent dry substance, 12.1 D.E., pH 5.2) were setup. The immobilized enzymes prepared as described above were introducedinto the reactors and stirring commenced. Periodically, the D.E.s of theconverted solutions were determined. After 70 hours, the contents ofeach of the reactors was filtered, the filter cakes washed extensivelywith deionized water and then added to 400 ml of partially hydrolyzedstarch solution (30.8 percent dry substance, 12.1 D.E., pH 5.2, 0.02 Min acetate buffer). Periodically, the D.E.s of the converted solutionswere determined. After 94 hours the contents of each of the reactorswere filtered, the filter cakes washed extensively with deionized waterand then added to 400 ml of partially hydrolyzed starch solution (30.8percent dry substance, 12.1 D.E., pH 5.2, 0.02 M in acetate buffer). TheD.E.s of the converted solutions were determined periodically. Theresults of these experiments are set forth in Tables IV to VII below:

                                      TABLE IV                                    __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase                                                      D. E. (Dextrose Equivalent)                            Immobilized Enzyme System                                                                            22 hrs.                                                                           46 hrs.                                                                           70 hrs.                                                                           94 hrs.                                    __________________________________________________________________________    10.7 g of immobilized glucoamylase                                            (first use) having a total activity                                           of 160.5 GU            70.3                                                                              77.8                                                                              79.8                                           Filter cake of immobilized glucoamylase                                       (second use) recovered from converted                                         solution above after 70 hours of use.                                                                74.6                                                                              78.8                                                                              80.9                                                                              86.5                                       Filter cake of immobilized glucoamylase                                       (third use) recovered from converted                                          solution above after 94 hours of use.                                                                74.8                                                                              82.4                                                                              84.9                                           __________________________________________________________________________

                                      TABLE V                                     __________________________________________________________________________    Saccharification Using Immmobilized Glucoamylase and Pullulanase                                     D.E. (Dextrose Equivalent)                             Immobilized Enzyme System                                                                            22 hrs.                                                                           46 hrs.                                                                           70 hrs.                                                                           94 hrs.                                    __________________________________________________________________________    10.7 g of immobilized                                                                    0.5 g of immobilized                                               glucoamylase having a                                                                    pullulanase having a                                               total activity of                                                                        total activity of                                                  160.5 GU (first use).                                                                    4 IU (first use).                                                                         80.5                                                                              85.1                                                                              87.1                                           Filter cake of immobilized glucoamylase                                       and pullulanase (second use) recovered from                                   converted solution above after 70 hours of use.                                                      80.0                                                                              84.4                                                                              86.2                                                                              87.7                                       Filter Cake of immobilized glucoamylase                                       and pullulanase (third use) recovered from                                    converted solution above after 94 hours of use.                                                      79.4                                                                              84.2                                                                              88.6                                           __________________________________________________________________________

                                      TABLE VI                                    __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase, Pullulanase                  and Saccharifying Alpha-Amylase                                                                                D.E. (Dextrose Equivalent)                   Immobilized Enzyme System        22 hrs.                                                                           46 hrs.                                                                           70 hrs.                                                                           94 hrs.                          __________________________________________________________________________    10.7 g of immobilized                                                                    0.5 g of immobilized                                                                    2.5 g of immobilized                                     glucoamylase having a                                                                    pullulanase having a                                                                    alpha-amylase having                                     total activity of                                                                        total activity of                                                                       a total activity of                                      160.5 GU.  4 IU.     18.5 liquefons.                                                                           96.9                                                                              96.8                                                                              95.8                                 Filter cake of immobilized glucoamylase, pullulanase and                      alpha-amylase (second use) recovered from converted solution -above after     70 hours of use.                 91.2                                                                              95.0                                                                              95.4                                                                              95.4                             Filter cake of immobilized glucoamylase, pullulanase and                      alpha-amylase (third use) recovered from converted solution                   above after 94 hours of use.     86.0                                                                              92.4                                                                              93.2                                 __________________________________________________________________________

                                      TABLE VII                                   __________________________________________________________________________    Saccharification Using Immobilized Glucoamylase                               and Saccharifying Alpha-Amylase                                                                        D. E. (Dextrose Equivalent)                          Immobilized Enzyme System                                                                              22 hrs.                                                                           46 hrs.                                                                           70 hrs.                                                                           94 hrs.                                  __________________________________________________________________________    10.7 g of immobilized                                                                     2.5 g of immobilized                                              glucoamylase having a                                                                     alpha-amylase having                                              total activity of                                                                         a total activity of 18.5                                          160.5 GU (first use).                                                                     liquefons (first use).                                                                     95.5                                                                              96.4                                                                              95.9                                         Filter cake of immobilized glucoamylase and alpha-                            amylase (second use) recovered from converted                                 solution above after 70 hours of use.                                                                  88.4                                                                              93.1                                                                              94.6                                                                              95.1                                     Filter cake of immobilized glucoamylase and alpha-                            amylase (third use) recovered from converted                                  solution above after 94 hours of use.                                                                  83.0                                                                              89.4                                                                              90.7                                         __________________________________________________________________________

What is claimed is:
 1. A process for converting starch to dextrosecomprising treating starch with alpha-amylase or acid or a combinationthereof to obtain a partially hydrolyzed starch solution containing atleast 10 percent hydrolyzed starch and then treating the partiallyhydrolyzed starch solution with an enzyme system comprising entrappedglucoamylase within a polymeric matrix and immobilized alpha-amylaseselected from the group consisting of alpha-amylase covalently bonded toan insoluble carrier, alpha-amylase adsorbed on an insoluble carrier,and entrapped alpha-amylase under conditions whereby a hydrolysatecontaining at least about 92% dextrose on an ash free, dry basis isproduced.
 2. A process for converting starch to dextrose as defined inclaim 1, wherein the alpha-amylase is entrapped in a polymeric matrix.3. A process for converting starch to dextrose comprising treatingstarch with alpha-amylase or acid or a combination thereof to obtain apartially hydrolyzed starch solution containing at least 10 percenthydrolyzed starch and then treating the partially hydrolyzed starchsolution with an enzyme system comprising entrapped alpha-amylase andimmobilized glucoamylase selected from the group consisting ofglucoamylase covalently bonded to an insoluble carrier and glucoamylaseadsorbed on an insoluble carrier.
 4. A process for converting starch todextrose as defined in claim 3, wherein the alpha-amylase is entrappedin a polymeric matrix.